Withaferin A activates TRIM16 for its anti-cancer activity in melanoma - PubMed (original) (raw)

Withaferin A activates TRIM16 for its anti-cancer activity in melanoma

Zsuzsanna Nagy et al. Sci Rep. 2020.

Abstract

Although selective BRAF inhibitors and novel immunotherapies have improved short-term treatment responses in metastatic melanoma patients, acquired resistance to these therapeutics still represent a major challenge in clinical practice. In this study, we evaluated the efficacy of Withaferin A (WFA), derived from the medicinal plant Withania Somnifera, as a novel therapeutic agent for the treatment of melanoma. WFA showed selective toxicity to melanoma cells compared to non-malignant cells. WFA induced apoptosis, significantly reduced cell proliferation and inhibited migration of melanoma cells. We identified that repression of the tumour suppressor TRIM16 diminished WFA cytotoxicity, suggesting that TRIM16 was in part responsible for the cytotoxic effects of WFA in melanoma cells. Together our data indicates that WFA has potent cytopathic effects on melanoma cells through TRIM16, suggesting a potential therapeutic application of WFA in the disease.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

WFA has selective toxicity to melanoma cells compared with fibroblast cell lines. (A), A panel of melanoma (MelJD, MelCV, G361, A375 and MM200) cells and normal fibroblasts (WI-38, MRC-5) were treated with increasing concentrations (0–5 μM) of Withaferin A (WFA) for 48 h and cell viability was measured using the Alamar Blue assay. (B), Average IC50 for WFA-treated melanoma cell lines and normal fibroblasts. Melanoma cell lines (MelJD and MelCV) and normal fibroblasts (WI-38, and MRC-5) were treated with increasing concentrations (0–5 μM) of WFA for 48 h, followed by (C), BrdU cell proliferation assay (D), MITOPROBE DILC1(5) measurements. (E), Flow cytometry results of MelJD and MelCV cells treated with increasing concentrations of WFA, then stained with Annexin-V-FITC/7AAD. Results are mean ± SEM, data was normalized to vehicle control treatment group.

Figure 2

Figure 2

WFA treatment decreases melanoma cell migration. MelJD and MelCV cells treated with indicated concentrations of WFA for 18 h. Results are mean ± SEM, data was normalized to vehicle control treatment group.

Figure 3

Figure 3

WFA treatment induces TRIM16 protein expression. (A), Western blot images of TRIM16 and GAPDH protein expression in MelJD and MelCV cells treated with WFA at increasing concentrations. The full-length blots are presented in the Supplementary Fig. 3A. (B), The level of TRIM16 protein expression measured by densitometry quantification. (C), RT-qPCR analysis of TRIM16 mRNA expression in MelJD and MelCV cells treated with WFA at increasing concentrations. Results are mean ± SEM, data was normalized to vehicle control treatment group.

Figure 4

Figure 4

Knock-down of TRIM16 protects melanoma cells against WFA. (A), Western blot analysis of TRIM16 expression in MelJD and MelCV cells following TRIM16 knock-down. GAPDH was used as internal control. The full-length blots are presented in the Supplementary Fig. 3B. MelJD and MelCV cells were transfected with control siRNA (siControl) or TRIM16 siRNAs (siTRIM16) for 24 h, then treated with WFA for additional 48 h followed by (B), Alamar Blue cell viability and (C), and BrdU cell proliferation measurements. Results are mean ± SEM, differences in cell viability and proliferation were compared to the vehicle (DMSO) treated siRNA control cells. (D), MelJD and MelCV cells expressing TRIM16 siRNAs (siTRIM16) or control siRNA (siControl) were seeded for colony formation assay and treated with indictaed concentrations of WFA for 72 h then allowed for colonies to form for 10 days, followed by crystal violet staining. (E), Quantification of colony formation assay based on crystal violet absorbance (590 nm). Differences in colony formation were compared to the vehicle treated control siRNA. MelJD and MelCV cells were transfected with control siRNA (siControl) or TRIM16 siRNAs (siTRIM16) for 24 h, then treated with WFA for additional 48 h followed by (F,) MITOPROBE DILC1(5) and (G), SYTOX Green measurements. Results are mean ± SEM, differences in cell viability and proliferation were compared to the vehicle control siRNA control cells.

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References

    1. Carlino MS, Long GV. Ipilimumab combined with nivolumab: A standard of care for the treatment of advanced melanoma? Clin. Cancer Res. 2016;22:3992–3998. doi: 10.1158/1078-0432.CCR-15-2944. - DOI - PubMed
    1. Long GV, et al. Dabrafenib plus trametinib versus dabrafenib monotherapy in patients with metastatic BRAF V600E/K-mutant melanoma: Long-term survival and safety analysis of a phase 3 study. Ann. Oncol. 2017;28:1631–1639. doi: 10.1093/annonc/mdx176. - DOI - PMC - PubMed
    1. Vanden Berghe W, Sabbe L, Kaileh M, Haegeman G, Heyninck K. Molecular insight in the multifunctional activities of Withaferin A. Biochem. Pharmacol. 2012;84:1282–1291. doi: 10.1016/j.bcp.2012.08.027. - DOI - PubMed
    1. Vyas AR, Singh SV. Molecular targets and mechanisms of cancer prevention and treatment by withaferin a, a naturally occurring steroidal lactone. AAPS J. 2014;16:1–10. doi: 10.1208/s12248-013-9531-1. - DOI - PMC - PubMed
    1. Bargagna-Mohan P, et al. The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin. Chem. Biol. 2007;14:623–634. doi: 10.1016/j.chembiol.2007.04.010. - DOI - PMC - PubMed

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